Neutrophil CD64 index as a good biomarker for early diagnosis of bacterial infection in pregnant women during the flu season

Abstract Background Pregnant women are at high risk of developing febrile illness during the flu season. Early identification of a viral or bacterial infection is crucial in the management of febrile pregnant patients. Neutrophil CD64 (nCD64) has been shown to have more important diagnostic value in sepsis than traditional inflammatory indicators. Methods The pregnant women enrolled were divided into three groups according to disease: influenza A infection, bacterial infection and healthy controls. Peripheral blood CD64, leukocyte, C‐reactive protein (CRP), procalcitonin (PCT) and human Th1/Th2‐related cytokines levels were routinely measured. The correlation between and diagnostic value of the nCD64 index and other biomarkers were evaluated using Spearman's correlation test and receiver operating characteristic (ROC) curve analysis. Results Pregnant women with bacterial infection had significantly elevated levels of leukocytes (8.4 vs. 5.95, 109/L; P = 0.004), CRP (89.70 vs. 50.05 mg/mL; P = 0.031), PCT (0.13 vs. 0.04 ng/mL; P = 0.010) and TNF‐α (0.46 vs. 0.38 pg/mL; P = 0.012) and an elevated nCD64 index (12.16 vs. 0.81; P < 0.001) compared with those with influenza A infection. The area under the receiver operating characteristic (AUROC) curve of the nCD64 index to discriminate bacterial infection among pregnant women (AUROC = 0.9183, P < 0.0001) was the largest. The sensitivity and specificity of the nCD64 index at an optimal cut‐off value of 3.16 were 84% and 100%, respectively, with a negative predictive value (NPV) of 94%. Conclusions Our study demonstrates the clinical value of the nCD64 index in distinguishing between bacterial infection and influenza A in pregnant women.


| INTRODUCTION
Pregnant women are a special group of people in terms of febrile diseases during the influenza season. Once infected with the influenza virus or bacteria, they have more than four times the hospitalization rates of the general population and are at increased risk of complications. 1,2 Pathogenic microbial infection is associated with adverse pregnancy outcomes. For example, pregnant women with influenza A accounted for 13% of all deaths during the 2009 US flu season, and Listeria monocytogenes infection caused 35% of foetal deaths in Israel. [1][2][3] Thus, early monitoring of inflammatory biomarkers is essential for febrile pregnant women.
However, traditional biomarkers of inflammation, such as C-reactive protein (CRP) level and leukocyte count, may make it difficult to identify the causes of inflammation. 4 Procalcitonin (PCT) is a diagnostic marker for sepsis in critically ill patients, 5 but is not effective for some local infections. In assessing bacterial coinfection with influenza, PCT was superior to CRP, and PCT < 0.29 ng/mL had a high negative predictive value (NPV) of 94% for excluding coinfection, especially in patients without shock. 6 Moreover, it has been reported that the inflammatory changes produced in peripheral blood leukocytes during normal pregnancy resemble those of sepsis due to the activation of the innate immune response. 7 Hence, identifying novel inflammatory biomarkers for the rapid distinction between bacterial infection and influenza in pregnant patients during the influenza season is crucial. CD64 (FcγRI) is the FcγR family member with the highest affinity for immunoglobulin G and is expressed on monocyte (Mo)-macrophages, neutrophils and eosinophils. 8,9 The FcγRI-antibody-pathogen complex is formed when the pathogenic organism is infected and mediates a series of downstream inflammatory immune responses.
Earlier studies have demonstrated the important diagnostic value of CD64 in the systemic inflammatory response. The CD64 index may play a supporting role in the diagnosis and treatment of neonatal sepsis and is considered to be a more reliable indicator for early diagnosis than leukocyte, CRP and PCT levels. 10,11 A meta-analysis of 14 studies of CD64 in septic patients concluded that neutrophil CD64 (nCD64) was an excellent biomarker for identifying sepsis in adult patients, with better accuracy than CRP and PCT levels. 12 In addition, nCD64 has been reported to be higher in patients with influenza A infection, both with and without pneumonia, than in healthy people. 13,14 A further study revealed that nCD64 could help differentiate between bacterial and viral infections. 15 The pregnant state is a delicate immunological balance in which maternal cells must prevent antigenic rejection of the foreign foetus while simultaneously maintaining adequate maternal defence functions to fight against infections. 9,16 A progressive upregulation of CD64 was detected in the peripheral circulation during pregnancy, supporting the idea that pregnancy leads to increased innate immunity. 17 Pregnant women with acute bacterial infection had higher levels of CD64 on the surface of neutrophils and Mos than women who were not pregnant. 18 However, little is known about the ability of the nCD64 index to discriminate between bacterial and viral infections in pregnant women.
In this study, we aimed to evaluate the practical value of the nCD64 index in distinguishing between bacterial infection and influenza A in pregnant women infected early in the influenza season. The flow chart and QUADAS-2 of this study are outlined in Figure 1 and Supplementary S1.

| Examination of traditional inflammatory indicators: leukocytes, CRP and PCT
The levels of leukocytes, CRP and PCT in the peripheral blood of patients were measured in a clinical laboratory at the Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine. Routine blood counts were performed with a Mindray CAL 8000 haematology analyser (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). CRP was measured by a particle-enhanced turbidimetric immunoassay. PCT was measured by a chemiluminescent immunoassay using an automatic chemiluminescence apparatus (Caris200) and diagnostic kits (Xiamen Innodx Biotechnology Co., Ltd.). All operations strictly complied with the experimental procedures provided by the manufacturers.

| Cytometric bead array to identify and determine human Th1/Th2-related cytokines
Peripheral blood samples were centrifuged for serum separation. Two millilitres of capture beads were suspended by vigorously vortexing for a few seconds before mixing. Then, a 300 μL aliquot of the capture beads from each assay tube to be analysed was added into a single tube labelled 'mixed capture beads'. The bead mixtures were incubated with the capture beads in serum enhancement buffer. The mixed capture beads were centrifuged at 200 Â g for 5 min.
The mixed capture bead pellet was resuspended in serum enhancement buffer. The mixed capture beads were incubated for 30 min at room temperature protected from light, and then 25 μL was added to all assay tubes. The BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit II (BD Biosciences, USA) was used to quantitatively measure interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) protein levels in a single sample. Human Th1/Th2 cytokine standard dilutions (25 μL) were added to the control assay tubes. The samples to be tested were added (25 μL) into the tubes. Next, 25 μL of Human Th1/Th2-II PE Detection Reagent was added to all assay tubes, and the samples were incubated for 2.5 h at room temperature protected from light. A 1 mL aliquot of wash buffer was added to each assay tube, and the tubes were then centrifuged at 200 Â g for 5 min. The supernatant from each assay tube was carefully aspirated and discarded. Finally, 100 μL of wash buffer was added to each assay tube to resuspend the bead pellet for testing.

| Statistical analysis
Statistical analysis was performed using SPSS for Windows release 19.0 (SPSS Inc., Chicago, IL, USA) for the data and GraphPad Prism 7 (GraphPad, Inc., CA, USA) for the graphs. Continuous variables are presented as medians with interquartile ranges (IQRs). The comparisons of continuous variables between independent groups were performed using the Mann-Whitney U test (two groups) or Kruskal-Wallis's test (multiple groups) followed by Dunn's post-test for F I G U R E 1 Flow chart for the admission of febrile pregnant women. multiple comparisons. The chi-square test was used for count data.
Spearman's correlation test was used to explore the correlation between the biomarkers. The sensitivity and specificity of the different indicators were compared by area under the receiver operating characteristic (AUROC) curve analysis. Optimal cut-off values to obtain the highest AUROC were calculated using the Youden index. A P-value <0.05 was considered a statistically significant difference between groups. There was no significant difference in age or gestational age among the three groups of pregnant women, as shown in Table 1.

| Comparison of traditional inflammatory biomarkers between pregnant women with influenza A and with bacterial infection
Pregnant women with bacterial infection had significantly elevated levels of blood leukocytes (P < 0.01) and serum levels of CRP (P < 0.05) and PCT (P < 0.05) compared with those with influenza A infection, as shown in Table 1   There was no difference in the blood levels of human Th1/Th2-related cytokines between healthy pregnant women and healthy nonpregnant women.

| AUROC of inflammatory biomarkers in pregnant women with infections (bacterial infection and influenza A)
To compare the value of different inflammatory biomarkers for identifying pregnant women with early infection during the influenza season, 30 pregnant women with bacterial infection and 24 pregnant women with influenza A were included for the AUROC of leukocyte, CRP, PCT, IL-6 and nCD64 index levels.
We found that the AUROC of the nCD64 index for discriminative ability in bacterial pregnant women (AUROC = 0.9183, P < 0.0001, 95% confidence interval (CI) 0.8364 to 1.000) was the largest and was significantly higher than that of leukocytes (AUROC = 0.7417, gestational age group. 21 A multicentre prospective observational study showed that a cut-off value of serum PCT > 0.25 ng/mL had a sensitivity of 87% and a specificity of 79% and was a better biomarker than CRP and leukocytes in differentiating acute pyelonephritis from asymptomatic bacteriuria and acute cystitis in pregnancy. 22 However, a recent study demonstrated that maternal serum CRP was a better predictive biomarker of histological chorioamnionitis than PCT. 23 In our study, the three traditional biomarkers (leukocytes, CRP and PCT) in the bacterial infection group were significantly higher than those in the influenza A group. Using receiver operating characteristic (ROC) curves, we found that leukocytes were a better bio- It has been reported that pregnant women infected with H1N1 influenza had significantly higher serum levels of IL-6, IL-10 and TNFα than healthy pregnant women, and IL-6 was correlated with the severity of the disease. 25,26 In our study, compared with healthy preg- Furthermore, elevated TNF-α is often associated with adverse pregnancy outcomes, 27 and in our study, almost all pregnant women had good deliveries at the follow-up visit, which may be related to maintaining the dynamic balance of TNF-α/IL-10. Many prospective studies have shown that the role of cytokines in the distinction between viral and bacterial pathogens is limited, including IL-6 and IFN-γ. 28 We reported similar findings in our study.
As an important Fc receptor mediating the inflammatory immune response, CD64 showed a biphasic expression pattern in peripheral blood, with an initial increase after 2 h, a more pronounced increase after 6 h and a decline within 48 h of stimulus removal. 29,30 In comparison with traditional biomarkers of inflammation, the nCD64 index has been found to be a relatively reliable biomarker of sepsis, 11,12 although reports on infection in pregnant women are scarce. A previous study suggested that the nCD64 index may not be a reliable indicator for distinguishing bacterial from viral infections. 4  and was significantly higher than that of leukocytes, PCT and CRP, whereas the AUROC of IL-6 was the smallest and showed no statistical significance. This provided the basis for the conclusion that nCD64 could be an optimal blood biomarker for the early diagnosis of bacterial infection in pregnant women during the influenza season.
The sensitivity and specificity of the nCD64 index at an optimal cut-off value of 3.16 were 84% and 100%, respectively, with an NPV of 94%. There are seldom researches on the application of the nCD64 index among the pregnant women with viral infections. We consider this test would be valid in other type of viral flu among the pregnant population. This is an area we need to validate further in the future.
There were several limitations to the study we conducted. First, this was a single-centre study, which could lead to selection bias. Second, the number of pregnant women enrolled was small, which might introduce a certain degree of statistical bias. Third, cases of influenza with bacterial infection were not included, making the value of the nCD64 index in this population unclear.
In summary, the differences in traditional indicators of inflammation (leukocytes, CRP and PCT), cytokines and the nCD64 index in the peripheral blood of pregnant women with bacterial infection or influenza A during the influenza season were investigated. These findings suggest that the nCD64 index could be an excellent biomarker for the early diagnosis of bacterial infection in pregnant women that is more reliable than leukocytes, CRP, PCT and IL-6. In the long term, we hope to conduct a multicentre study and include cases of influenza coinfection with bacteria to better understand the value of the nCD64 index in pregnant women with infections.